Development of a nanopore technology has been performed popularly. “Advanced Sequencing Technology Awards” which started to be granted by NHGRI (National Human Genome Research Institute, U.S.A.) in 2004 by using decoding the human genome in 2003 as a meter, was established in order to recommend technological development of a next-generation sequencer. The largest investment of these grants is the nanopore technology. The nanopore technology occupies 29 of 60 projects to which funds were granted from 2004 to 2010.
The nanopore includes bionanopore and solid nanopore. The bionanopore treats protein and a lipid bilayer which are biomolecules. These substances are easily denatured disadvantageously. When measurement is performed with the bionanopore, it is necessary to supply protein and the lipid bilayer to a flow cell before the measurement, and workability is poor. Therefore, expectation for the solid nanopore which does not have to treat these biomolecules has been increased.
One of big problems of the solid nanopore technology is that a passing speed of a DNA molecule passing through the nanopore is too high. In the present circumstances, the speed of DNA passing through the solid nanopore is 100 μm/s (=3 μs/base). It is necessary to reduce the passing speed to 0.3 μm/s (=1 ms/base) in order to detect a base sequence with resolution of one base. This problem is reported in detail in NPL 1.
As a method for reducing the passing speed of DNA, in NPL 2, a method for adjusting the temperature, pH, viscosity, or the like of a solution, is examined. Fologea et al. have succeeded in reducing the passing speed of a DNA molecule from 0.3 m/s to 1 mm/s by optimizing these conditions. However, 1 mm/s is higher than 0.3 μm/s as an ideal speed by about four orders of magnitude, and it is necessary to further reduce the passing speed. It is recognized that a signal of a blockage current used for detecting a DNA base is also reduced disadvantageously with the increase of the passing speed.
PTL 1 proposes a nanopore film having a film structure in which a plurality of conductive films and insulating films are stacked on each other. It is described that the passing speed of a DNA molecule passing through a nanopore can be controlled by using this nanopore film. A cylindrical piezoelectric element is embedded in a nanopore opening. The piezoelectric element is in contact with the conductive film to be energized. The piezoelectric element can be extended or compressed by a nanometer scale according to a voltage applied thereto. A DNA molecule in a solution is guided to the nanopore opening by an electrode disposed in a flow cell, invades the nanopore opening, and starts to pass through the nanopore. The time when the DNA molecule starts to pass through the nanopore can be specified by detecting decrease in a blockage current. At the same time as the time when the DNA molecule starts to pass through the nanopore, a voltage is applied to the piezoelectric element embedded in the nanopore opening, and the piezoelectric element is expanded. The DNA molecule in the nanopore can be thereby captured. It is possible to control the passing speed of the DNA molecule in the nanopore by controlling a potential with respect to the piezoelectric element in a pulse while an external electric field is applied to the DNA molecule. Alternatively, it is described that each base of the DNA molecule can go unidirectionally in the nanopore.
PTL 2 proposes a nanopore structure in which a gate electrode film is sandwiched by insulating films and the outside thereof is further sandwiched by a source electrode film and a drain electrode film. This patent literature is also for controlling a passing speed of a DNA molecule in a nanopore. It is possible to control an orientation of ions in a solution including an electrolyte in the nanopore by further applying a gate voltage while a constant voltage is applied between the source and drain electrode films in advance. For example, it is possible to form a (negative, positive, negative) ion layer in the nanopore while the gate voltage is applied. On the other hand, it is possible to form a (negative, negative, negative) ion layer in the nanopore while the gate voltage is not applied. In the former state, flows of the ions are blocked in two regions separated by a nanopore film. However, in the latter state, the ions can flow in. This means that an ion current in an aqueous solution can be controlled. MOS-FET which is popular in a digital camera or the like controls an electronic circuit by turning on or off a flow of electrons. Similarly to this, a flow of ions in the aqueous solution can be controlled in this patent literature. In this patent literature, this idea is named “nano fluidic FET.” The DNA molecule and the ions are charged. In a broad sense, motion of DNA in the nanopore can be regarded as a flow of ions. Therefore, it is possible to control the passing speed of the DNA molecule in the nanopore using the “nano fluidic FET”.
In PTL 3, a nanopore film is produced using an insulating film. An organic molecule is modified to a nanopore opening in contact with DNA. A weak and transitional bond (for example, hydrogen bond) is formed between this organic molecule and the DNA molecule. This bond is stronger than thermal fluctuation of the DNA molecule, and therefore the DNA molecule can be captured in the nanopore. It is described that a passing speed of DNA can be controlled by applying a voltage to the DNA molecule in a pulse in vertical upper and lower directions of the nanopore film.
In conventional nanopore measurement, a phenomenon in which an ion current is reduced when the DNA molecule passes through the nanopore, that is, a blockage current is measured. However, the blockage current is extremely feeble, and parallelization is difficult. Therefore, a method in which a microelectrode is disposed on the nanopore film and a tunneling current generated when the DNA passes is measured, is becoming the mainstream. In NPL 3, it was confirmed that monomers A, T, G, C, and U had electric conductivities different from each other in measurement of a tunneling current using a microelectrode. Thereafter, it was tried to discriminate one base in a three base DNA (GGG, GTG, TGT, or the like) and a seven base RNA (UGA GGU A, apart of cancer marker let-7 miRNA sequence). In the change of the current with time, a step-shaped change for each base was observed. However, the change was not observed in the order of the sequence, but some parts of the sequence, such as GGTG or TG, were repeated or missed. This is because the moving direction of the DNA molecule cannot be controlled and the DNA molecule moves at random by an irregular Brownian motion. When only the step-shaped change of a current was extracted and all the data points converted into the electric conductivity were made into a histogram, a distribution having two peaks was observed. A value of the electric conductivity of the peak value was the same as a value obtained by the monomer. It was also possible to measure the change of the electric conductivity for each base of the seven base RNA. It was found that the seven base sequence could be determined by arranging the above-described measurement sequences in a large amount like a shotgun sequence and performing statistical processing.
Meanwhile, it is a thermal ratchet mode that has attracted attention as a method for moving a substance unidirectionally in a micro region. This model includes a ratchet having asymmetric teeth, an impeller, and a clasp on the wall. This impeller is in a gas at a temperature T1. Therefore, a gas molecule collides with the impeller, and the impeller receives a random force clockwise or counterclockwise. The temperature of the clasp is represented by T2. A spring is attached to the clasp. When the ratchet tries to rotate clockwise, the clasp retracts to the left side and the ratchet can rotate. When the ratchet tries to rotate counterclockwise, the ratchet stops at the clasp. In this system, it is known that unidirectional rotation does not occur as long as there is no temperature difference between the temperature T1 of the impeller and the temperature T2 of the spring. In other words, it is known in principle that unidirectional motion is caused if a temperature difference between T1 and T2 can be introduced. NPL 4 describes this technology.
A single stranded DNA molecule is a polymer obtained by polymerizing molecules dATP, dCTP, dGTP, and dTTP which are asymmetric and have very similar structures. Therefore, the single strand DNA molecule has a spiral shape, and an asymmetric shape and a potential for each base on an axis thereof. Conventionally, an elastic rod model which assumes symmetry of a molecule has been employed for rigidity of the DNA molecule. However, experimental results in which the rigidity of the DNA molecule cannot be explained with this model have been submitted recently. In order to explain this, NPL 5 describes the rigidity of the DNA molecule with an asymmetric elastic rod model which assumes asymmetry of the DNA molecule.
As a material of the nanopore, graphene has attracted attention recently. Graphene is obtained by extending a hexagonal frame formed by carbon into a sheet-shape. Graphene is obtained by extracting one atomic surface of a graphite crystal. At present, measurement of a blockage current is employed most often in reading a base of DNA in the nanopore. When DNA passes through a nanopore, an effective area of the nanopore through which ions can pass changes. Therefore, a current flowing in upper and lower spaces of the nanopore also changes. This changing current is referred to as the blockage current. This blockage current is measured, and discrimination of four bases included in DNA is performed. However, when a film of the nanopore is too thick, several nucleotide molecules are contained in the film thickness. Therefore, as a result, it is difficult to correspond a base sequence for each base in the DNA molecule to the measured blockage current. A distance between the nucleotides in the DNA molecule is from 0.32 to 0.52 nm. It is necessary to introduce a film having a thickness almost the same as this distance in order to discriminate the base sequence of a DNA molecule passing through the nanopore. Graphene is a useful material to solve this problem. NPL 6 reports that resolution of graphene having a thickness of 0.6 nm for discriminating the DNA base is 0.35 nm in computer simulation. NPL 7 reports excellent physical properties of graphene. The thermal conductivity of graphene is highest in the currently known substances, and is 5000 [W/m/K]. On the other hand, the thermal conductivity of water is 0.6. There is a difference of about four orders of magnitude as the order of thermal conductivity. The Young's modulus of graphene is highest in the currently known substances, and is 1500 [GPa].